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Melon regeneration in vitro method and application of melon regeneration in vitro method in melon genetic transformation

An in vitro regeneration and melon technology, applied in the biological field, can solve the problems of poor reproducibility of the regeneration system, low differentiation rate of direct differentiation of adventitious buds, difficulty in callus differentiation and budding, etc.

Active Publication Date: 2015-12-30
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
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  • Application Information

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Problems solved by technology

At present, there have been many successful reports on the genetic transformation of muskmelon through the organ differentiation pathway. However, in the process of in vitro regeneration of cotyledons, it is also difficult to differentiate and sprout from callus, and the rate of direct differentiation of adventitious buds is low. Therefore, the establishment of an efficient and stable regeneration system is still the focus of research

Method used

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  • Melon regeneration in vitro method and application of melon regeneration in vitro method in melon genetic transformation
  • Melon regeneration in vitro method and application of melon regeneration in vitro method in melon genetic transformation
  • Melon regeneration in vitro method and application of melon regeneration in vitro method in melon genetic transformation

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Embodiment 1

[0095] Embodiment 1, the method for in vitro regeneration of muskmelon

[0096] The method of muskmelon regeneration in vitro is as follows:

[0097] 1. Germination of seeds

[0098] The experiment was repeated three times, and the steps for each repetition were as follows:

[0099] (1) Seed disinfection: pick 30 plump melon (K7-1, K7-2, Topmark or Emerald) seeds, peel off the seed coat, soak in 70% (volume ratio) ethanol aqueous solution for 20-30s, and use Bacteria washed 2 to 3 times. Then the melon seeds were sterilized with 1% (mass to volume ratio) sodium hypochlorite aqueous solution for 10 minutes, washed with sterile water for 4 to 5 times, and blotted to dry excess water with sterile filter paper. (2) Place the sterilized seeds on MS solid medium at 28°C (26°C to 30°C in practical applications), culture in dark for 2 days, and then count the seed germination rate, seed germination rate = number of germinated seeds / sowing Number of seeds x 100%.

[0100] Accordi...

Embodiment 2

[0136] Embodiment 2, the application of the method for the in vitro regeneration of muskmelon according to embodiment 1 in establishing the genetic transformation method of muskmelon

[0137] 1. Construction of recombinant Agrobacterium EHA105 / pCHF3-Pm-2F

[0138] 1. Using the double-stranded DNA molecule shown in sequence 1 in the artificially synthesized sequence list as a template, use the artificially synthesized primer PchAF: 5-TC GAGCTC GATGGCAGAATCAATTCTG-3' (the underlined part is the SacI restriction site) and primer PchAR: 5'-CA GGATCC GTTAGTTCATGGTGGGAAGC-3' (the underlined part is the BamHI restriction site) was amplified by PCR to obtain a PCR amplified product and recover it.

[0139] 25μL PCR reaction system includes 50ng template DNA, 2.5μL 10×Buffer (containing 15mM MgCl 2 ), 1.0 μL dNTPs (2.5 mM concentration), 1UTaq DNA polymerase, 1 μL primer PchAF (10 μM), 1 μL primer PchAR (10 μM) and ddH 2 O. Among them, TaqDNA polymerase, 10×Buffer (containing 15m...

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Abstract

The invention discloses a melon regeneration in vitro method. The melon regeneration in vitro method provided by the invention comprises the following steps: (1) taking a cotyledon of a melon as an explant, inoculating the explant into a shoot induction medium for culturing to obtain an adventitious bud tussock; (2) placing the adventitious bud tussock obtained in the step (1) to a shoot elongation medium A for culturing to obtain an adventitious bud; (3) placing the adventitious bud obtained in the step (2) into a rooting medium for culturing to obtain a melon regeneration seedling. The melon regeneration in vitro method provided by the invention has the advantages of high adventitious bud inductivity, low vitrification degree, short regeneration period and the like, and by applying the method in the melon genetic transformation, a relatively high transformation rate can be obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for in vitro regeneration of muskmelon and its application in muskmelon genetic transformation. Background technique [0002] Melon (Cucumismelo L.) is an important fruit-type vegetable crop, belonging to Cucurbitaceae (Cucurbitaceae), genus Cucumis (Cucumis), a species of melon. Melon is widely cultivated at home and abroad. In recent years, with the large-scale development of melon production, the occurrence of melon diseases and insect pests has become more and more serious, resulting in a sharp decrease in the yield and quality of melon. [0003] Directed improvement of germplasm by means of transgenic technology is an effective way to cultivate new varieties of muskmelon with high yield and high quality. Carrying out research on muskmelon transgenic technology is of great significance for the control of muskmelon pests and diseases and the improvement of varieties. Co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H4/00
Inventor 许勇张春秋张海英宫国义
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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